Schematic illustration and characterization of B16 tumor cells expressing differentially localized antigen.

<p>(A) Schematic depiction of gene constructs used to generate B16 parental (B16.par) tumors expressing EGFP-CL4 recombinant protein in secretory (B16.Sec), nuclear (B16.Nuc), or cytoplasmic (B16.Cyto) cellular compartments. (B) Confocal fluorescence microscopy demonstrated targeted expression of EGFP-CL4 in distinct subcellular compartments within respective tumors (magnification, ×60). (C) Quantitative imaging flow cytometry confirmed confocal microscopy observations. (D) EGFP ELISA was used to compare the amount of EGFP present in whole cell lysates (W.C.L) and supernatants (Sup.) of respective tumors. A standard curve of recombinant EGFP protein was used to calculate the amount of EGFP. A representative experiment is shown out of two independent ELISAs in triplicates ± SEM. (E) Relative intensity and stability of EGFP expression was determined by live-cell flow cytometry. Mean fluorescent intensities (MFI) are shown in figure. (F) <i>In vivo</i> growth pattern of respective tumors after s.c. inoculation in F1 mice. Data are means of 10 mice from two independent experiments. ***p<0.001. One-way ANOVA, followed by a Bonferroni post-test.